In the development stage of a nerve, a dynamic structure, a so-called growth cone, is formed at a tip of an axon of the nerve as the axon extends to a target tissue. The growth cone detects signaling molecules therearound, and changes its extension direction in response to diffusible or contact-dependent guidance cues. For the detection and so forth, complex systems including organelles and receptors are utilized (NPL 1). Most of axon guidance factors identified until today are proteins and derivatives thereof. However, the researches on the lipid-based mechanism have revealed the presence of lysophosphatidic acids (NPLs 2, 3), sphingosine-1-phosphate (NPL 4), and endocannabinoids (NPL 5) as molecules playing a role in signal transduction in the brain.
As the interface between the central nerve and the peripheral nerve, accurate circuit formation in the spinal cord is important for neural development. Particularly, a connection between a dorsal root ganglion (DRG) sensory afferent axon and a spinal cord second-order neuron is a crucially important step. There are different subtypes of DRG sensory afferent nerve, which are projected into various regions in the spinal cord. All of these must enter the spinal cord via a limited region of the dorsal white matter, that is, a dorsal root entry zone (DREZ), and extend axons thereof to an appropriate target site probably while directed to a guidance cue. DRG sensory nerves with an afferent axon projected into the spinal cord are classified into two groups according to the neurotrophin receptor expression. Specifically, one is a TrkA receptor-expressing neuron dependent on NGF, a ligand of the receptor; the other is a TrkC receptor-expressing neuron dependent on NT-3, a ligand of the receptor (NPL 6). Moreover, main TrkA-expressing nerves are nociceptive, whose afferent axon terminates at the outermost layer of the dorsal horn. Meanwhile, most of TrkC-expressing nerves are proprioceptive or mechanoreceptive, whose axon is projected into a deep portion of the ventral gray matter (NPL 7).
To date, various molecular mechanisms have been identified, which control pattern formation of primary DRG afferent nerve in the spinal cord. For example, in the chicken spinal cord, axonin/TAG-1 and F11 are necessary to correctly guide nociceptive and proprioceptive afferent nerves, respectively (NPL 8). Further, in mice, a transient domain of dorsally derived netrin-1 plays an important role in controlling a timing when to enter the gray matter at an interstitial branching of collateral (NPL 9). Nevertheless, researches are most concentrated on semaphorin 3A (Sema3A). Semaphorin 3A has been identified as collapsin (factor suppressing projection extension) in the early development of avians (NPL 10), and is involved in different pattern formations of nociceptive and proprioceptive afferent nerves in the spinal cord. Specifically, it has been revealed that the former is inhibited or repelled by secreted semaphorin 3A, whereas the latter is non-responsive to the repellent (chemorepellent) signal (NPLs 11 to 15). Such a difference in chemical reactivity between the two types of nerve groups is adjusted by dynamic expression of neuropilin-1 (NRP-1), a semaphorin signal receptor. Specifically, as development proceeds, the region where Sema3A is expressed is gradually restricted to the ventral spinal cord; simultaneously, the expression of NRP-1 is increased in a nociceptive nerve. In addition, the expression of NRP-1 in proprioceptive neurons is correspondingly decreased (NPLs 15, 16).
As described above, various researches have been conducted on semaphorin 3A, and a lot of findings have been obtained. Meanwhile, the presence of a sensory nerve-guiding mechanism independent of semaphorin signal transduction in the spinal cord has been suggested for a long period of time based on in vitro and in vivo experiments (NPL 17). Tissue sections collected from the ventral spinal cord of mice homozygously deficient for Sema3A or wild type mice demonstrate a repellent effect on DRG axon extension in a coculture assay using a collagen gel (NPL 18). Moreover, in a case of in vivo, most of TrkA and TrkC nerves of Sema3A-deficient mice exhibit normal central projections into targets in ventral and dorsal gray matters (NPLs 19 to 21). Further, although an in vivo analysis cannot be conducted on a projection in the gray matter at a late stage of development because most of NRP-1-deficient mice die at embryonic day 12, the spinal cord-afferent projection is normal at least at an early stage of development (NPL 22). However, the result of a culture experiment on tissue sections collected from the ventral spinal cord of NRP-1-deficient mice confirmed a repellent effect on a DRG axon in vitro (NPL 18). Moreover, cells of double NRP-1 and Nrp-2 knockout mice are, in theory, insensitive to signal transduction by diffusible class 3 semaphorin, but die at embryonic day 8 before such an analysis can be conducted because the blood circulatory system is defective (NPL 23).
Sharma and Frank performed a microsurgical manipulation on chicken such that the ventral spinal cord of chicken was replaced with the dorsal spinal cord located opposite thereto, followed by in vitro culturing. In this event, since a sample derived from a chicken deficient in the ventral spinal cord also still had stereotypic pattern formations of proprioceptive and nociceptive afferent neurons, Sharma and Frank contest a ventral-dorsal concentration gradient mechanism of a long-range diffusible repulsive factor (NPL 24). Nonetheless, it has been revealed that before the interstitial collateral extends into the gray matter, Sema3A signal transduction plays a role in the early pattern formation of a DRG afferent nerve in DREZ in vivo. Furthermore, Gu and associates produced viable NRP-1 mutant mice by a genetic approach, and the DRG afferent nerve showed precocious interstitial extension into the dorsal horn (NPL 25). In addition, Bron and colleagues obtained a similar result by targeting SiRNA to NRP-1 in a developing spinal cord in a chicken embryo by employing in ovo electropolation. Specifically, it is known that a DRG axon “temporarily stops” the extension at a stage when the DRG axon reaches the DREZ of the spinal cord before reaching the dorsal horn. However, it was revealed that when SiRNA knocks down NRP-1, such “temporary stop (waiting period)” is shortened, and the afferent nerve prematurely enters the gray matter, extends straight as it is and reaches the midline (NPL 26). Nevertheless, such a research outcome suggests that unlike a proposed differentiation mechanism at a late stage of development, defects in the early pattern formation cause premature inward extension of TrkA and TrkC afferent nerves. Moreover, inappropriate extensions are more frequently observed in the result of the above chicken embryos than in the result of the viable knockout mice produced by Gu and colleagues. It is pointed out that this is based on a fundamental difference in guiding mechanism between rodents and avians. In mice, inward extensions of proprioceptive afferent and nociceptive afferent nerves are continuous, and the inward extension of the latter precedes that of the former by at least 24 hours. On the other hand, in chickens, both occur simultaneously. This suggests that regarding avians, the guidance cue be more important than the developmental timing to distinguish regions of the two afferent sensory nerves (NPL 27).
Meanwhile, phosphatidylglucoside (PtdGlc), one of membrane glycolipid molecules, has been identified in Staphylococcus aureus (NPL 28). Further, recently, similar lipids have been identified in mammalian cells as putative intercellular signal transducers (NPLs 29, 30). Then, a PtdGlc-specific monoclonal antibody (DIM2 antibody) was produced (NPL 31), and PtdGlc was identified as a marker of radial glial cells in the cortex of rat (NPL 32). It is known that PtdGlc is localized in most of the CNS (central nervous system) including the spinal cord, in addition to the cortex (NPL 32). Furthermore, lysophosphatidylglucoside is a hydrolysate of PtdGlc, and reported to demonstrate a strong repellent effect on axon extension (PTL 1). A molecule capable of suppressing the activity of a molecule having such a repellent effect, if developed, can promote repairing of a neural circuit in nervous system disorders, neurodegenerative disorders, and neuronal damages. However, such a molecule is yet to be developed.